DA-9801, a standardized Dioscorea extract, improves memory function via the activation of nerve growth factor-mediated signaling.

OBJECTIVES: Nerve growth factor (NGF) is a neurotrophin that plays a critical role in mammalian learning and memory functions. NGF also regulates neuronal cell differentiation and neurite outgrowth by activating ERK/CREB signaling. This present study examined the effects of a standardized Dioscorea extract (DA-9801), which is composed of Dioscorea japonica Thunb and Dioscorea nipponica Makino on memory function via its NGF-potentiating activities using an in vitro and in vivo paradigm. METHODS: Cells were incubated with or without different concentrations of DA-9801 (10, 25, and 50 µg/ml) extract for 24 h. The cultured conditioned medium from C6 glioma cells was used for NGF production assay, and neurite length in N2a cells was measured after every 2 h. Mice were orally treated with DA-9801 (10 and 100 mg/kg/day) once daily for 7 days. They were subjected to passive avoidance test to evaluate memory functions. The question of whether DA-9801 induced NGF synthesis was assessed by measuring the levels of NGF in the mouse cortical and hippocampal tissues. Hippocampal cell differentiation and NGF-mediated ERK/CREB signaling were evaluated by performing immunohistochemical analysis using BrdU, ki67, DCX, phosphorylated ERK and CREB in the mouse hippocampus. RESULTS: DA-9801 treatment increased the NGF contents and neurite length, respectively. Mice with DA-9801 administration showed memory enhancement in the passive avoidance test. DA-9801 also increased newborn cell differentiation, neurite length, NGF secretion, and ERK/CREB phosphorylation in the mouse hippocampus. DISCUSSION: These results suggest that DA-9801 treatment could improve memory function by inducing hippocampal NGF synthesis and ERK/CREB signaling.

Novel Botanical Therapeutic NB-02 Effectively Treats Alzheimer's Neuropathophysiology in an APP/PS1 Mouse Model.

Alzheimer's disease (AD) is an incurable neurodegenerative disorder and a major cause of dementia. Some of the hallmarks of AD include presence of amyloid plaques in brain parenchyma, calcium dysregulation within individual neurons, and neuroinflammation. A promising therapeutic would reverse or stymie these pathophysiologies in an animal model of AD. We tested the effect of NB-02, previously known as DA-9803, a novel multimodal therapeutic, on amyloid deposition, neuronal calcium regulation and neuroinflammation in 8- to 10-month-old APP/PS1 mice, an animal model of AD. In vivo multiphoton microscopy revealed that two-month-long administration of NB-02 halted amyloid plaque deposition and cleared amyloid in the cortex. Postmortem analysis verified NB-02-dependent decrease in plaque deposition in the cortex as well as hippocampus. Furthermore, drug treatment reversed neuronal calcium elevations, thus restoring neuronal function. Finally, NB-02 restored spine density and transformed the morphology of astrocytes as well as microglia to a more phagocytic state, affecting neuroinflammation. NB-02 was effective at reversing AD neuropathophysiology in an animal model. Therefore, in addition to serving as a promising preventative agent, NB-02 holds potential as a treatment for AD in the clinic.

Novel botanical drug DA-9803 prevents deficits in Alzheimer's mouse models.

BACKGROUND: Alzheimer's disease (AD) is a neurodegenerative disorder characterized by deposition of amyloid plaques and disruption of neural circuitry, leading to cognitive decline. Animal models of AD deposit senile plaques and exhibit structural and functional deficits in neurons and neural networks. An effective treatment would prevent or restore these deficits, including calcium dyshomeostasis observed with in-vivo imaging. METHODS: We examined the effects of DA-9803, a multimodal botanical drug, in 5XFAD and APP/PS1 transgenic mice which underwent daily oral treatment with 30 or 100 mg/kg DA-9803 or vehicle alone. Behavioral testing and longitudinal imaging of amyloid deposits and intracellular calcium in neurons with multiphoton microscopy was performed. RESULTS: Chronic administration of DA-9803 restored behavioral deficits in 5XFAD mice and reduced amyloid-ß levels. DA-9803 also prevented progressive amyloid plaque deposition in APP/PS1 mice. Elevated calcium, detected in a subset of neurons before the treatment, was restored and served as a functional indicator of treatment efficacy in addition to the behavioral readout. In contrast, mice treated with vehicle alone continued to progressively accumulate amyloid plaques and calcium overload. CONCLUSIONS: In summary, treatment with DA-9803 prevented structural and functional outcome measures in mouse models of AD. Thus, DA-9803 shows promise as a novel therapeutic approach for Alzheimer's disease.

Combined Application of UHPLC-QTOF/MS, HPLC-ELSD and 1 H-NMR Spectroscopy for Quality Assessment of DA-9801, A Standardised Dioscorea Extract.

INTRODUCTION: DA-9801, a standardised 50% aqueous ethanolic extract of a mixture of Dioscorea japonica and D. nipponica, is a botanical drug candidate for the treatment of diabetic neuropathy, which finished its US phase II clinical trials recently. An advanced quality control method is needed for further development of DA-9801, considering its high contents of both primary and secondary metabolites. OBJECTIVE: Development of a quality assessment strategy for DA-9801, based on the combination of UHPLC-QTOF/MS, HPLC-ELSD, and 1 H-NMR spectroscopy. METHODS: The method was developed and tested with 15 batch products of DA-9801. The steroidal saponins of DA-9801 were tentatively identified by UHPLC-QTOF/MS and were quantified with the validated HPLC-ELSD method. Primary metabolites of DA-9801 were identified and profiled using 1 H-NMR spectrometry. The batch-to-batch equivalence of DA-9801 was tested with the 1 H-NMR spectra using spectral binning, correlation analysis, and principal component analysis. RESULTS: Six major saponins of DA-9801 were tentatively identified by UHPLC-QTOF/MS. Among them, protodioscin and dioscin were quantified by the validated HPLC-ELSD method. Twenty-six metabolites were identified in 1 H-NMR spectra. The similarity between DA-9801 batches could be evaluated with the NMR spectra of DA-9801. The 1 H-NMR method also revealed that two Dioscorea species contributed distinct amino acids to the contents of DA-9801. CONCLUSION: This study validates the effectiveness of UHPLC-QTOF/MS, HPLC-ELSD, and 1 H NMR-combined method for quality control of DA-9801 and its crude materials. Copyright © 2016 John Wiley & Sons, Ltd.

Odisolane, a Novel Oxolane Derivative, and Antiangiogenic Constituents from the Fruits of Mulberry (Morus alba L.).

Mulberry, the fruit of Morus alba L., is known as an edible fruit and commonly used in Chinese medicines as a warming agent and as a sedative, tonic, laxative, odontalgic, expectorant, anthelmintic, and emetic. Systemic investigation of the chemical constituents of M. alba fruits led to the identification of a novel oxolane derivative, (R*)-2-((2S*,3R*)-tetrahydro-2-hydroxy-2-methylfuran-3-yl)propanoic acid (1), namely, odisolane, along with five known heterocyclic compounds (2-6). The structure of the new compound was elucidated on the basis of HR-MS, 1D and 2D NMR ((1)H-(1)H COSY, HSQC, HMBC, and NOESY) data analysis. Compound 1 has a novel skeleton that consists of 8 carbon units with an oxolane ring, which until now has never been identified in natural products. The isolated compounds were subjected to several activity tests to verify their biological function. Among them, compounds 1, 3, and 5 significantly inhibited cord formation in HUVECs. The action mechanism of compound 3, which had the strongest antiangiogenic activity, was mediated by decreasing VEGF, p-Akt, and p-ERK protein expression. These results suggest that compounds isolated from M. alba fruits might be beneficial in antiangiogenesis therapy for cancer treatment.

DA-9801 promotes neurite outgrowth via ERK1/2-CREB pathway in PC12 cells.

In the present study, we examined the mechanisms underlying the effect of DA-9801 on neurite outgrowth. We found that DA-9801 elicits its effects via the mitogen-activated protein kinase (MEK) extracellular signal-regulated kinase (ERK)1/2-cAMP response element-binding protein (CREB) pathway. DA-9801, an extract from a mixture of Dioscorea japonica and Dioscorea nipponica, was reported to promote neurite outgrowth in PC12 cells. The effects of DA-9801 on cell viability and expression of neuronal markers were evaluated in PC12 cells. To investigate DA-9801 action, specific inhibitors targeting the ERK signaling cascade were used. No cytotoxicity was observed in PC12 cells at DA-9801 concentrations of less than 30 µg/mL. In the presence of nerve growth factor (NGF, 2 ng/mL), DA-9801 promoted neurite outgrowth and increased the relative mRNA levels of neurofilament-L (NF-L), a marker of neuronal differentiation. The Raf-1 inhibitor GW5074 and MEK inhibitor PD98059 significantly attenuated DA-9801-induced neurite outgrowth. Additionally, the MEK1 and MEK2 inhibitor SL327 significantly attenuated the increase in the percentage of neurite-bearing PC12 cells induced by DA-9801 treatment. Conversely, the selective p38 mitogen-activated protein kinase inhibitor SB203580 did not attenuate the DA-9801 treatment-induced increase in the percentage of neurite-bearing PC12 cells. DA-9801 enhanced the phosphorylation of ERK1/2 and CREB in PC12 cells incubated with and without NGF. Pretreatment with PD98059 blocked the DA-9801-induced phosphorylation of ERK1/2 and CREB. In conclusion, DA-9801 induces neurite outgrowth by affecting the ERK1/2-CREB signaling pathway. Insights into the mechanism underlying this effect of DA-9801 may suggest novel potential strategies for the treatment of peripheral neuropathy.

Evaluation of the transporter-mediated herb-drug interaction potential of DA-9801, a standardized dioscorea extract for diabetic neuropathy, in human in vitro and rat in vivo.

BACKGROUND: Drug transporters play important roles in the absorption, distribution, and elimination of drugs and thereby, modulate drug efficacy and toxicity. With a growing use of poly pharmacy, concurrent administration of herbal extracts that modulate transporter activities with drugs can cause serious adverse reactions. Therefore, prediction and evaluation of drug-drug interaction potential is important in the clinic and in the drug development process. DA-9801, comprising a mixed extract of Dioscoreae rhizoma and Dioscorea nipponica Makino, is a new standardized extract currently being evaluated for diabetic peripheral neuropathy in a phase II clinical study. METHOD: The inhibitory effects of DA-9801 on the transport functions of organic cation transporter (OCT)1, OCT2, organic anion transporter (OAT)1, OAT3, organic anion transporting polypeptide (OATP)1B1, OATP1B3, P-glycoprotein (P-gp), and breast cancer resistance protein (BCRP) were investigated in HEK293 or LLC-PK1 cells. The effects of DA-9801 on the pharmacokinetics of relevant substrate drugs of these transporters were also examined in vivo in rats. RESULTS: DA-9801 inhibited the in vitro transport activities of OCT1, OCT2, OAT3, and OATP1B1, with IC50 values of 106, 174, 48.1, and 273 µg/mL, respectively, while the other transporters were not inhibited by 300 µg/mL DA-9801. To investigate whether this inhibitory effect of DA-9801 on OCT1, OCT2, and OAT3 could change the pharmacokinetics of their substrates in vivo, we measured the pharmacokinetics of cimetidine, a substrate for OCT1, OCT2, and OAT3, and of furosemide, a substrate for OAT1 and OAT3, by co-administration of DA-9801 at a single oral dose of 1,000 mg/kg. Pre-dose of DA-9801 5 min or 2 h prior to cimetidine administration decreased the Cmax of cimetidine in rats. However, DA-9801 did not affect the elimination parameters such as half-life, clearance, or amount excreted in the urine, suggesting that it did not inhibit elimination process of cimetidine, which is governed by OCT1, OCT2, and OAT3. Moreover, DA-9801 did not affect the pharmacokinetic characteristics of furosemide, as evidenced by its unchanged pharmacokinetic parameters. CONCLUSION: Inhibitory effects of DA-9801 on OCT1, OCT2, and OAT3 observed in vitro may not necessarily translate into in vivo herb-drug interactions in rats even at its maximum effective dose.

Evaluation of DA-9801, a new herbal drug for diabetic neuropathy, on metabolism-mediated interaction.

DA-9801, the mixture extract of Dioscoreae rhizoma and Dioscorea nipponica Makino, is a new herbal drug currently being evaluated in a phase II clinical study for the treatment of diabetic peripheral neuropathy in Korea. The inhibitory potentials of DA-9801, D. rhizoma extract, D. nipponica Makino extract, and dioscin, an active component of DA-9801, on eight human cytochrome P450 (CYP) enzymes and four UDP-glucuronosyltransferase (UGT) enzymes were investigated in human liver microsomes using liquid chromatography-tandem mass spectrometry. DA-9801 showed slight inhibition of CYP1A2, CYP2C8, UGT1A1, and UGT1A9 enzyme activities with IC(50) values of 396.4, 449.9, 226.0, and 408.8 µg/mL, respectively. D. rhizoma extract showed negligible inhibition of CYP and UGT activities, but D. nipponica extract slightly inhibited CYP1A2, CYP2C8, CYP2C9, UGT1A1, and UGT1A9 activities with IC(50) values of 264.2, 237.1, 206.8, 302.4, and 383.1 µg/mL, respectively. DA-9801 showed volume per dose index values of 0.44-0.88 L for a 200-mg dose, suggesting that they may not cause the inhibition of the metabolism of CYP1A2, CYP2C8, UGT1A1, and UGT1A9-catalyzed drugs in humans. These results suggest that the administration of DA-9801 in human may not cause clinically relevant inhibition of these enzymes.

Neurotrophic activity of DA-9801, a mixture extract of Dioscorea japonica Thunb. and Dioscorea nipponica Makino, in vitro.

ETHNOPHARMACOLOGICAL RELEVANCE: Dioscorea japonica Thunb. has been traditionally used to treat polyuria and diabetes in Korea. AIM OF THE STUDY: We previously report the effects of Dioscorea japonica Thunb. extract on glucose control, NGF induction, and neuroprotection in a rodent diabetic model. Since the most potent fraction, DA-9801, was identified from a mixture of Dioscorea japonica Thunb. (DJ) and Dioscorea nipponica Makino (DN) following bioactivity-guided fractionation, here, we investigated the potential mechanism of the extract activity against diabetic peripheral neuropathy (DPN). MATERIALS AND METHODS: A 1:3 mixture of DJ and DN was extracted with ethanol (DA-9801) and further fractionated into an ethylacetate-soluble fraction (DA-9801E). Effects of these extracts on neurite outgrowth were measured in PC-12 cells and DRG neurons. Effects on cell viability and TrkA phosphorylation were evaluated in PC-12 cells. NGF induction effect was determined in primary Schwann cells as well as IMS32 cells (immortalized Schwann cells). RESULTS: No cytotoxicity was observed in PC-12 cells at the concentration below 500 µg/ml of either DA-9801 or DA-9801E. DA-9801 and DA-9801E at 100 µg/ml and 10 µg/ml, respectively, showed a significant effect on neurite outgrowth in PC-12 cells and DRG neurons in the presence of or absence a low concentration of NGF (2 ng/ml). The Trk-A phosphorylation effect of DA9801 was confirmed in PC-12 cells. An NGF induction effect of these extracts was not detected in either IMS-32 cells, or primary Schwann cells. CONCLUSIONS: The NGF agonistic activity of DA-9801 and DA-9801E was demonstrated, which may contribute to their neuroprotective effect against DPN. Studies of the detailed mechanism of these extracts as well as identification of the active components are warranted for the development of an anti-DPN drug from DJ and DN.

Novel botanical drug for the treatment of diabetic neuropathy.

Diabetic neuropathy has multiple aspects of pathophysiology including metabolic derangements and alterations of microvessel with hyperglycemia. Currently, there is no effective long-term treatment based on the pathologic mechanisms. Nerve regeneration with nerve growth factor (NGF) has been on clinical trials as a treatment option. This article reviews an action of a herbal medicine, DA 9801, whose mechanism of action is related to NGF in treating diabetic neuropathy.

Induction of pacemaker currents by DA-9701, a prokinetic agent, in interstitial cells of Cajal from murine small intestine.

The interstitial cells of Cajal (ICC) are pacemaking cells required for gastrointestinal motility. The possibility of whether DA-9701, a novel prokinetic agent formulated with Pharbitis Semen and Corydalis Tuber, modulates pacemaker activities in the ICC was tested using the whole cell patch clamp technique. DA-9701 produced membrane depolarization and increased tonic inward pacemaker currents in the voltage-clamp mode. The application of flufenamic acid, a non-selective cation channel blocker, but not niflumic acid, abolished the generation of pacemaker currents induced by DA-9701. Pretreatment with a Ca2+-free solution and thapsigargin, a Ca2+-ATPase inhibitor in the endoplasmic reticulum, abolished the generation of pacemaker currents. In addition, the tonic inward currents were inhibited by U-73122, an active phospholipase C inhibitor, but not by GDP-beta-S, which permanently binds G-binding proteins. Furthermore, the protein kinase C inhibitors, chelerythrine and calphostin C, did not block the DA-9701-induced pacemaker currents. These results suggest that DA-9701 might affect gastrointestinal motility by the modulation of pacemaker activity in the ICC, and the activation is associated with the non-selective cationic channels via external Ca2+ influx, phospholipase C activation, and Ca2+ release from internal storage in a G protein-independent and protein kinase C-independent manner.

Blockade of atopic dermatitis-like skin lesions by DA-9102, a natural medicine isolated from Actinidia arguta, in the Mg-deficiency induced dermatitis model of hairless rats.

DA-9102 isolated from Actinidia arguta is a candidate of natural medicine currently under Phase II clinical trial for atopic dermatitis in Korea. In this study, spontaneous dermatitis was induced by magnesium deficiency in hairless rats and this system was applied to assess the suppressive effects of DA-9102 on atopic dermatitis-like skin disease. Oral administration of DA-9102 at a dose of 100 mg/kg for 16 days substantially suppressed the occurrence of spontaneous dermatitis. Eczematous skin lesions, water loss and scratching behavior were significantly decreased by DA-9102 in a dose-dependent manner. Infiltration of inflammatory cells into the skin and pathologic remodeling of the epidermis and dermis were much less than the Mg-def. group. Results from flow cytometry analysis of peripheral blood mononuclear cells indicated that DA-9102 suppressed activation of leukocytes. The decrease in the number of CD45RA+ cells was accompanied by a lower level of IgE in DA-9102 treated rats, and the reduction in the number of CD11b+ cells by DA-9102 in both periphery and skin was significant. Further, DA-9102 not only suppressed the mRNA expression of T(H)2 cytokines including IL-4 and IL-10 in the lymph node but it also decreased the levels of inflammatory mediators such as nitric oxide and leukotriene B(4) (LTB(4)) in the serum. Taken together, these results suggest that DA-9102 is an orally applicable potent immune modulator capable of controlling the occurrence of atopic dermatitis-like skin disease.